Research On Function Of A Novel Matrix Protein PfY2 And Identification Of Nacreous Protein Complex

Pinctada fucata is an important economical seawater pearl production species.The formation of its nacre and shell has always been the research hotspot in biomineralization.The matrix proteins secreted by mantle epithelium play a very important regulatory role during this process.This study mainly focused on the novel matrix protein PfY2 and EDTA soluble nacreous matrix protein complex to explore their effects on the regulation of the formation shell formation.PfY2,a basic matrix protein,located on the EDTA-solube fraction from both prismatic and nacreous layer was identified by our group based on the gene expression profile of different larval developmental stages.RT-PCR results showed that it was highly expressed in both mantle edge and pallial,consistent with its location in shell components.After specifically knocking down its expression,both the prismatic and nacreous layer exhibited excessive growth to some extent.So we suspected that PfY2might inhibit the deposition of calcium carbonate crystals.Subsequently,we used the E.coli prokaryotic expression system to express and purify the recombinant protein r PfY2.After confirming its specific binding ability to calcite and aragonite,the in vitro crystallization experiments of Ca CO₃ crystals were applied to simulate the effects on shell formation.It was found that r PfY2 could participate in crystal nucleation,inhibit the crystallization rate and crystal growth,and also significantly prevent the transformation of amorphous calcium carbonate ACC to more stable crystallines,including calcite and aragonite.Based on the above results,we believe that this novel matrix protein PfY2 may act as a negative regulator and responsible for maintaining shell biomineralization balance.In addition,for the first time,we confirmed that at least one soluble protein complex was formed in nacreous layer with the help of BN-PAGE.Mass spectrometry identification indicated that this complex was consisted of 26 different matrix proteins.Co-immunoprecipitation and in vitro protein pull down assay confirmed the interaction between ESN1 and ESN2,the top two proteins ranked by Mascot in LC-MS.On the other hand,also for the first time,we purified the recombinant matrix protein r ESN1and r ESN2 from renaturation of bacterial inclusion bodies,which still held the specific binding ability to Ca CO₃ crystals and were also co-localized in calcite and aragonite.r ESN1 and r ESN2 participated in Ca CO₃ growth by stabilizing vaterite and inducing the formation of aragonite crystals only with 5 m M Mg²⁺.In conclusion,matrix protein PfY2 and soluble nacreous protein complex are all involved in the regulation of biomineralization,and further exploration of their working mechanism will improve the construction of matrix protein regulatory network during shell formation at the molecular level.